Field of the Invention
The present invention is in the field of stabilizing biological materials in a glassy dry structure.
Related Art
The preservation of the structure and function of biological materials during long-term storage at high temperature and humidity is of fundamental importance to the food, nutraceutical and pharmaceutical industries. Sensitive biological materials, such as proteins, enzymes, cells, bacteria and viruses must often be preserved for long-term storage for later use. Simple freezing is often done when drying is either harmful or unsuitable in the final product. For preservation in a dry state—freeze-drying has traditionally been the most common method. Other methods, such as ambient air-drying, drying under vacuum at ambient temperatures (vacuum-drying), or drying by contacting a fine mist of droplets with warm air (spray-drying) and drying by desiccation are generally not suitable for sensitive bioactives, such as live or attenuated bacteria and viruses. The high drying temperatures used in these methods result in significant damage to the bioactive itself.
Often the freeze drying process may result in a significant loss of activity and damage to the bioactive agent due to the formation of ice crystal during the slow drying process. Freeze-drying combines the stresses due to both freezing and drying. The freezing step of this process can have undesirable effects, such as the denaturation of proteins and enzymes, and rupture of cells. Damage caused by freezing may be circumvented, to a certain degree, by the addition of cryoprotective compounds or agents to the solution. Such protective agents are generally highly soluble chemicals that are added to a formulation to protect cell membranes and proteins during freezing and to enhance stability during storage. Common stabilizers include sugars such as sucrose, trehalose, glycerol, or sorbitol, at high concentrations (Morgan et al., 2006; Capela et al., 2006). Disaccharides, such as sucrose and trehalose, are natural cryoprotectants with good protective properties. Trehalose is a particularly attractive cryoprotectant because it has actually been isolated from plants and live organisms that remain in a state of suspended animation during periods of drought. Trehalose has been shown to be an effective protectant for a variety of biological materials, (see Crowe, J. H., 1983). Several patents disclose the use of trehalose or trehalose in combination with other cryoprotectants for protecting proteins and other biological macromolecules, such as enzymes, serum, serum complement, antibodies, antigens, fluorescent proteins and vaccine components during freezing, drying and rehydration (U.S. Pat. No. 5,556,771).
However, there are some drawbacks associated with the use of trehalose or other disaccharides or monosaccharides as the sole cryoprotectant. Trehalose may not penetrate adequately into the cell to protect active components within the intracellular volume, which may lead to instability upon storage of the freeze-dried substances. In addition, concentrations of trehalose greater than 60% by weight of a given preservation medium are sometimes necessary. An even more serious problem associated with the use of trehalose is that biological materials preserved using trehalose alone are not storage stable for extended periods of time, especially those stored at high temperatures and/or humid environments. Therefore, a significant challenge remains to develop an optimal formulation and drying process that minimizes drying losses while achieving adequate storage stability of the dried material.
Some of the issues associated with the trehalose and the freeze-drying process have been resolved by using a combination of certain formulations and vacuum drying in a glassy state, particularly sugar glasses (U.S. Pat. No. 6,190,701). In those formulations, the bioactive agent is protected within a glassy matrix against hostile environments such as high temperatures and humidity. However, in these formulations, the presence of water as moisture in the environment acts as a plasticizing agent and has the effect of lowering the glass transition temperature (Tg) of the glassy matrix. At higher water contents, the Tg is significantly lowered to the extent that the dry formulation is in the undesirable rubbery or plastic state at room temperature.
The advantages of retaining the glass form of the formulation include increased physical stability of the solid and reduction of deleterious intermolecular reactions. A detailed discussion of the physical chemistry of water-food polymer interactions as relating to the glassy state and their transition temperatures can be found in M. Le Meste, et al. 2002. However, limitations of amorphous systems such as physical instability and higher chemical reactivity, act as a hurdle in their extensive commercialization.
Thus, a need exists for a stabilizing composition that is useful for wide range biological materials. A further need exists for a stabilizing composition that can be effectively used in both freeze-drying processes and drying processes involving ambient-temperature drying. There is also a need for a composition mixture that is less costly than those presently being used. Finally, and importantly, there is a need for a composition mixture that provides stable media for preservation of biological materials over extended periods of time at elevated temperatures and varying degrees of humidity which can be encountered during shipping and storage of materials, while still retaining a significant amount of activity upon rehydration.
All of these needs are met by the composition mixture, drying methods and resulting preserved biological material compositions of the present invention.